Fifty years ago Haldene proposed the hypothesis that new mutation account for about one-third of cases of hemophilia and other X-linked "genetic lethal" conditions. He also stated that in a stationary population mutations to hemophilia would have a life of two to four generations. We now have the opportunity to determine directly the accuracy of Haldane's theory. In this proposal we will screen by DNA analysis about 500 hemophilia A families, and we expect to characterize the mutant allele of the factor VIII:C gene in about 100 of these families. (Parenthetically, Haldane postulated the existence of just two hemophilia alleles in 1935). We believe this is possible because we have found the mutation in 16 of 80 mutant genes screened to date. Among the 100 alleles we will discover that 50 of these mutations occurrred de novo within two generations of the proband. We will then determine 1) the distribution of de novo mutations in these tow generations, 2) the types of mutations (point mutations vs. deletions) and their sex distribution, and 3) whether advanced paternal (or maternal) age is associated with various types of new mutations. In characterizing the molecular defect in many of these mutant factor VIII:C genes, we expect to gain significant new insights into structure-function relationship in the factor VIII:C protein. In addition, we will quantify the extent to which CpG dinucleotides are mutation sensitive and discover whether particular CpG residues are more sensitive to mutation than others. Preliminary data indicate that this should be a highly useful approach because CG-TG and CG-CA mutations have now been observed by us and Gitschier et al in seven mutant alleles at 5 CpG sites studied in exon of the factor VIII:C gene. Two instances of recurrent CGA-TGA nonsenses mutations have been found which account for four alleles among the 80 mutant genes studied by us. In summary, the work proposed should provide definitive answers to a number of improtant questions involving the generation of mutations in man.